Pseudomonas aeruginosa is a Gram-negative bacterial pathogen of humans that can acutely infect susceptible patients to cause invasive, tissue-destructive infections, as well as chronically colonize patients with underlying lung disease, such as individuals with Cystic Fibrosis. Two polar surface organelles of P. aeruginosa-Type IV pili and flagella-play important roles in host-pathogen and pathogen-pathogen interactions required for infection and colonization. Both surface organelles are required for the initiation and development of biofilms, the structured multicellular bacterial communities that allow P. aeruginosa to persistently colonize abiotic and biotic environments, including that of the cystic fibrosis airway. We have characterized a P. aeruginosa protein, FimX, which is required for pilus assembly, adherence to and virulence toward host cells, and anaerobic biofilm formation. FimX is a phosphodiesterase for bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP), a second messenger implicated in the post-transcriptional control of many bacterial systems relevant to motility, colonization and virulence. Genomics has revealed over 2000 bacterial proteins containing domains predicted to synthesize or breakdown c-di-GMP, as well as proteins predicted to bind c-di-GMP and transduce this signal into specific phenotypes. However, our understanding of how individual diguanylate cyclases, c-di-GMP phosphodiesterases and c-di-GMP binding proteins interact to bring about specific bacterial behaviors is quite primitive. In this application, we will focus on FimX and a group of proteins that interact with it in the regulation of pilus assembly. We are particularly interested in examining how the protein-protein interactions that we have uncovered regulate and confer specificity upon c-di-GMP signaling by (1) altering the activity of c-di-GMP metabolizing enzymes in response to environmental cues and by (2) changing the subcellular location at which c-di-GMP is produced or degraded. By completing the experiments outlined in this proposal, we will expand our understanding of how P. aeruginosa regulates the assembly of pili and flagella during motility and biofilm formation, behaviors that are crucial to bacterial colonization and persistence during pathogenesis. However this work has broader implications as well, as it will allow us to address general questions relevant to c-di-GMP dependent signaling pathways in all prokaryotes. Relevance: The treatment of P. aeruginosa infections is often difficult: these bacteria are highly resistant to most antibiotics and grow as biofilms that cannot be effectively eradicated by antimicrobials or the human immune response. Completion of these experiments will advance us toward our long-term goal of understanding how environmental cues direct P. aeruginosa behaviors that enable it to colonize and infect human hosts. We anticipate that this knowledge will allow us to develop therapeutic agents that can disrupt these adaptive behaviors and limit the ability of Pseudomonas to cause human disease.